Background: It is known that cellular senescence could affect culture results. A previous study on K19 found that the loss of stem cell proportion is the reason for difficulties experienced when culturing aged keratinocytes. But the situation is unclear, because K19 is not generally accepted as an epidermal stem cell marker.
Objective: The aim of this study was to investigate the effects of cellular senescence caused by chronological aging or by repeated subcultures.
Methods: The effects of cellular senescence were investigated using monolayer cultures of keratinocytes and reconstructed epidermis. We prepared keratinocytes from donors of different ages and by repeated subcultures. Flow cytometric analysis was performed using alpha6 integrin and CD71 antibodies, and candidate keratinocyte stem cell proportions were separated according to reactivities to these antibodies. Living skin equivalents (LSEs) were reconstructed using keratinocytes from child, adult and elderly donors.
Results: Flow cytometric analysis showed a decrease in the candidate stem cell proportion in an age- or culture passage-dependent manner. LSE experiments showed that a reconstructed epidermis using child's keratinocytes was well formed compared to epidermis reconstructed using an elderly donor's keratinocytes. Different expression of proliferation markers was also observed according to donor age.
Conclusion: Our results showed that cellular senescence by chronological aging or repeated sub-culture induced the loss of candidate stem cell proportion in keratinocyte cultures. This seems to be the reason why it is difficult to culture keratinocytes from the elderly or by repeatedly culturing keratinocytes in vitro.